14350-14355, 2005). In the work described here, we show that the E295K gastric carcinoma pol ␤ variant acts in a dominant-negative manner by interfering with base excision repair. This leads to an increase in sister chromatid exchanges. Expression of the E295K variant also induces cellular transformation. Our data suggest that unfilled gaps are channeled into a homology-directed repair pathway that could lead to genomic instability. The results indicate that base excision repair is critical for maintaining genome stability and could therefore be a tumor suppressor mechanism.

Approximately 30% of human tumors characterized to date express DNA polymerase beta (pol β) variant proteins. Two of the polymerase beta cancer-associated variants are sequence-specific mutators, and one of them binds to DNA but has no polymerase activity. The Leu22Pro (L22P) DNA polymerase beta variant was identified in a gastric carcinoma. Leu22 resides within the 8 kDa amino terminal domain of DNA polymerase beta, which exhibits dRP lyase activity. This domain catalyzes the removal of deoxyribose phosphate during short patch base excision repair. We show that this cancer-associated variant has very little dRP lyase activity but retains its polymerase activity. Although residue 22 has no direct contact with the DNA, we report here that the L22P variant has reduced DNA-binding affinity. The L22P variant protein is deficient in base excision repair. Molecular dynamics calculations suggest that alteration of Leu22 to Pro changes the local packing, the loop connecting helices 1 and 2 and the overall juxtaposition of the helices within the N-terminal domain. This in turn affects the shape of the binding pocket that is required for efficient dRP lyase catalysis.

The ⑀ subunit of Escherichia coli DNA polymerase III possesses 3-exonucleolytic proofreading activity. Within the Pol III core, ⑀ is tightly bound between the ␣ subunit (DNA polymerase) and subunit. Here, we present the crystal structure of ⑀ in complex with HOT, the bacteriophage P1-encoded homolog of , at 2.1 Å resolution. The ⑀-HOT interface is defined by two areas of contact: an interaction of the previously unstructured N terminus of HOT with an edge of the ⑀ central ␤-sheet as well as interactions between HOT and the catalytically important helix ␣1-loop-helix ␣2 motif of ⑀. This structure provides insight into how HOT and, by implication, may stabilize the ⑀ subunit, thus promoting efficient proofreading during chromosomal replication.The precise mechanisms by which cells are able to duplicate their DNA with both high accuracy (Ͻ1 error/10 10 bases replicated) and speed (up to 1000 nucleotides/s) are of major interest. Chromosomal replication is performed by multisubunit replicases that conduct the simultaneous, coordinated replication of the leading and lagging strands. Among the model systems currently being investigated, the best understood is that of the bacterium Escherichia coli (1, 2). In this organism, chromosomal replication is performed by DNA polymerase III holoenzyme (HE) 2 , a dimeric complex containing 10 distinct subunits (17 total). Within the HE complex (␣⑀) 2 ␤ 4 2 (␥␦␦Ј), there are two polymerase core assemblies (␣⑀), one for each strand, which are the primary determinants of replication fidelity. Each core consists of the ␣ subunit (the polymerase, (M r ϭ 135,000)), the ⑀ subunit (a 3Ј 3 5Ј exonuclease that acts as a proofreader for polymerase misinsertion errors, (M r ϭ 28,500)), and the subunit (M r ϭ 8,000), connected in the linear order ␣-⑀-.The ⑀ subunit, encoded by the dnaQ gene, plays a critical role within the Pol III core, both catalytically and structurally. Many dnaQ mutants exhibit strong mutator phenotypes, whereas a fully catalytically deficient mutation causes lethality due to excessively high mutation rates (error catastrophe) (3). Deletion mutants of dnaQ have been generated, but they also proved to be nonviable unless accompanied by a suppressing mutation in the polymerase (4). Based on these studies, ⑀ is thought to have at least two functions: a fidelity function and a structural function, due to its tight and presumably stabilizing interaction with the polymerase. Recently, a potential second proofreading activity has been discovered residing in the N-terminal PHP domain of the ␣ subunit (5, 6), which also contains the binding site for ⑀. Interaction between ⑀ and ␣ is dependent on the C-terminal domain of ⑀ (residues 187-243) (7,8). In contrast, the N-terminal domain of ⑀ (residues 1-186) contains the exonuclease active site and retains binding affinity for the subunit.The subunit does not have any known enzymatic function; its role within the Pol III core is presumed to be structural, through its interaction with the ⑀ subunit. Strains lacking (⌬holE mutants), al...